Peripheral Cellular Immune Responses Induced by Subretinal Adeno-Associated Virus Gene Transfer Can Be Restrained by the Subretinal-Associated Immune Inhibition Mechanism.

After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.109 vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.

Subretinal Injection of HY Peptides Induces Systemic Antigen-Specific Inhibition of Effector CD4+ and CD8+ T-Cell Responses.

Purpose: Injection of an antigen into the anterior chamber of the eye induces a peripheral antigen-specific immune modulation mechanism, known as anterior chamber-associated immune deviation (ACAID). Delayed-type hypersensitivity experiments argue that the subretinal space (SR) of the eye displays properties similar to ACAID. However, no investigation was performed regarding the differential impact of a subretinal antigen injection on peripheral CD4+ versus CD8+ T cells, on the potential immune deviation regarding Th profiles, and on the antigen-specificity of the inhibition. A better understanding of these mechanisms is crucial to improve safety and immunomonitoring of ongoing therapeutic approaches targeting the SR. The aim of this study is to characterize the proliferative capacities and cytokine patterns of antigen-specific CD4+ and CD8+ T cells after a subretinal injection of antigen in mice. Methods: Ubiquitously Transcribed tetratricopeptide repeat gene Y-linked (UTY) and DEAD Box polypeptide 3 Y-linked (DBY) peptides which respectively include MHCI- and MHCII-restricted T-cell epitopes of the mouse HY male antigen, were injected into the subretinal space of C57BL/6 female mice. 2 weeks later, these mice were immunized subcutaneously with these peptides and compared to control mice. A week later, T-cell immune responses were analyzed by IFNγ ELISpot assays and cytokine measurements (IL-2, IL-4, IL-6, IL-10, IL-13, IL-17a, IFNγ, TNFα, GM-CSF, and MCP-1) in the spleen and with proliferation assays in draining lymph nodes. Results: Immune cells from mice that received HY peptides in the SR before immunization, compared with those from control immunized mice, secreted significantly smaller quantities of Th1/Tc1, Th2/Tc2, and Th17/Tc17 cytokines, and HY-specific CD4+ T cells proliferated less in response to HY peptides. Conclusion: Taken together, our data clearly demonstrate that the subretinal injection of HY peptides induces a systemic HY-specific inhibition of conventional Th profiles and CD8+ T cells. We propose to call this phenomenon SRAII, for subretinal-associated immune inhibition.

Cellular and Molecular Mechanisms of Anterior Chamber-Associated Immune Deviation (ACAID): What We Have Learned from Knockout Mice.

Anterior chamber-associated immune deviation (ACAID) is a well-known phenomenon that can occur after an antigen is introduced without any danger signal into the anterior chamber of a murine eye. It is reported to lead to an antigen-specific immune deviation throughout the body. Despite the relatively little evidence of this phenomenon in humans, it has been suggested as a potential prophylactic strategy in allograft rejections and in several autoimmune diseases. Cellular and molecular mechanisms of ACAID have been explored in different murine models mainly as proofs of concept, first by direct analyses of immune components in normal immunocompetent settings and by cell transfer experiments. Later, use of knockout (KO) mice has helped considerably to decipher ACAID mechanisms. However, several factors raise questions about the reliability and validity of studies using KO murine models. This mini-review summarizes results obtained with KO mice and discusses their advantages, their potential weaknesses, and their potential methods for further progress.